Method for Preparing FMOC-Based Hydrolysable Linkers

ABSTRACT

A novel process for the production of Fmoc (9H-fluoren-9-ylmethoxycarbonyl)-based compounds is provided, wherein a protecting group for the 9-hydroxymethyl group of the fluorene ring system is utilized. These compounds are useful for the modification of protein and peptide drugs.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority to U.S. provisionalapplication Ser. No. 60/937,125, filed Jun. 26, 2007, which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the preparation of a hydrolysablelinker, which is useful for the modification of protein and peptidedrugs.

BACKGROUND OF THE INVENTION

Most peptide and protein drugs are short-lived and have often a shortcirculatory half-life in vivo. Considering that peptide and proteindrugs are not absorbed orally, prolonged maintenance of therapeuticallyactive drugs in the circulation is a desirable feature of obviousclinical importance.

An attractive strategy for improving clinical properties of protein orpeptide drugs is a modification of protein or peptide drugs withpolymers e.g. polyalkylene-oxides (Roberts et al., Advan Drug Rev. 54,459-476 (2002)) or polysaccharides like Polysialic acid (Fernandes etal., Biochim Biophys Acta 1341, 26-34 (1997)), dextranes orhydroxylethyl starch. The modification with poly(ethylene glycol) (PEG)has been known for a while. However, modification of proteins with PEGoften leads to reduction of the activity of the protein. Thereforealternative systems were developed allowing the releasable coupling ofthe polymer to the protein or peptide drug using hydrolysable ordegradable chemical linkers (U.S. Pat. No. 6,515,100, U.S. Pat. No.7,122,189, WO 04/089280, WO 06/138572). The protein-polymer conjugatecan be regarded as a prodrug and the activity of the protein can bereleased from the conjugate via a controlled release mechanism. Usingthis concepts improved pharmacokinetic properties of the drug can beobtained (Zhao et al., Bioconjugate Chem. 17, 341-351 (2006)).

Therefore, WO 04/089280 suggested the use of a hydrolysable PEG-linker.(All documents cited in the specification are incorporated byreference.)

Tsubery et al., (J Biol Chem. 279, 38118-38124 (2004)) demonstrated ahydrolysable PEG-linker for derivatization of proteins based on the Fmoc(9-fluorenylmethyl carbamate) group. A fluorene group is reacted withmaleimidopropionic anhydride and N-hydroxysuccinimide, which is furtherreacted with poly(ethylene glycol) (PEG) and proteins by their aminogroups. However, the synthesis of the hydrolysable linker, namedMAL-FMS-OSU (9-Hydroxymethyl-2-(amino-3-maleimido-propionate)-7-sulfofluorene N-hydroxysuccinimidyl carbonate), suffers from low yield andreduced reproducibility. The key problem with the synthesis according toTsubery et al. is the introduction of the maleimide group by reaction of9-Hydroxymethyl-2-amino fluorene with maleimido propionic acidanhydride. In this step undesired side reactions like esterification ofthe OH group in position 9 occurred. Thus, an improved synthesiscontaining an additional step for protection of the OH group wasdeveloped.

SUMMARY OF THE INVENTION

The present invention provides a new method for the synthesis of acompound of general formula 1:

wherein PG is a protecting group and at least one of position 1, 2, 3,4, 5, 6, 7 or 8 is bound to radical Y.

Y is a radical containing a N-maleimidyl-moiety.

In addition to being bound to radical Y the compound of formula 1 mayoptionally be bound to radical X in at least one of the availableposition 1, 2, 3, 4, 5, 6, 7 or 8.

X is —SO₃—R³.

R³ is selected from the group consisting of hydrogen, (C₁-C₈)-alkyl and(C₁-C₈)-alkyl-R⁴.

R⁴ is a polymer.

Compounds of the invention are prepared according to a multi-stepprotocol wherein a protecting group for the 9-hydroxymethyl group of thefluorene ring system is being utilized. These derivatives can be furthermodified to yield an activated ester such as an succinimidyl ester.

DETAILED DESCRIPTION OF THE INVENTION

The present invention allows to overcome the above problems by asynthesis which utilizes a protecting group to yield a compound offormula 1. A compound of formula 1 is a suitable precursor forsubsequent reaction steps yielding a hydrolysable linker, likeMAL-FMS-OSU and other MAL-Fmoc-OSU-derivatives containing an activemaleimide (MAL) and a N-hydroxysuccinimide (OSU=NHS) group and providesthe desired products in high yield and purity. These linkers can befurther modified with one or more polymers and can then be used tomodify a peptide or protein drug.

The compounds of formula 1 are prepared starting from anamino-substituted fluorene. The new synthetic protocol introduces aprotecting group for the hydroxylmethyl group at the 9 position of thefluorene in step 4 of the protocol shown below. The synthetic schemebelow illustrates the preparation of a compound of formula 1 as anexample:

Further Reaction Steps:

Step 1:

In a first step (see scheme above), an amine group of anamino-substituted fluorene is protected by a BOC group(tert-Butyloxycarbonyl), for example by reaction with BOC-anhydride orthe like. Any other suitable protecting group (Greene et el., ProtectiveGroups in organic synthesis, Jon Wiley & Sons, Inc., Third Edition, NewYork (1999)) for amines can be used. An additional example is the Z(Benzyloxycarbonyl) group.

Multi-amino-substituted fluorene derivatives can be used in a similarreaction in order to synthesize a compound of formula 1 having more thanone radical Y in position 1, 2, 3, 4, 5, 6, 7 or 8.

Step 2:

In a second step a hydroxymethyl group is introduced at position 9 ofthe fluorene core, for example, by reaction with NaH orLithiumdiisopropylamid (LDA) and ethylformiate and subsequent reactionwith NaBH₄ or other reductive agents like DIBAL (Diisobutylaluminiumhydride) in MeOH.

Step 3:

In a third step the BOC protecting group is cleaved, for example, withHCl CF₃COOH or p-toluolsulfonic acid.

Step 4:

In a fourth step the 9-hydroxymethyl group is protected, for example, byreaction with a silyl halogenide such as tBDMS-Cl (Corey et al., J AmChem Soc. 94, 6190-6191 (1972)) or 4,4′-Trimethoxytritylchlorid.

in one embodiment the silyl halogenide is tBDMS-Cl(tert-butyldimethylsilyl chloride). Preferably, the reaction withtBDMS-Cl is performed with imidazole in DMF (dimethylformamide). The useof a silyl protecting group makes the molecule more lipophilic, thusfacilitating the preparation of compounds, which are bound to more thanone radical Y.

Step 5:

In a fifth step a N-maleimidyl-moiety is introduced, for example, byreacting the amino group with a maleimidoalkylic acid or amaleimidoalkylic acid anhydride.

The maleimidyl group is reactive towards thiole groups. Therefore,modified polymers such as PEG-SH can be covalently bound to thehydrolysable linker to yield a polymer-modified hydrolysable linker.

Step 6:

In this optional step, radical X (—SO₃R³) is introduced in the fluorenering system. This acidic group makes the compound more hydrophilic andallows to perform subsequent coupling reactions in aqueous solvents. Inaddition a sulfonic acid group allows the coupling of a second polymerby esterification with a OH-group of a polymer.

This procedure allows to introduce radical X to yield a compound offormula 1, comprising in addition to radical Y in position 1, 2, 3, 4,5, 6, 7 or 8 also radical X in at least one of the available position 1,2, 3, 4, 5, 6, 7 or 8.

In one embodiment, at least one radical X is bound to positions 2, 4, 5and/or 7. In another embodiment, radical X is bound to position 7.

In another embodiment radical X (—SO₃R³) is introduced in the fluorenering system after step 4. If —SO₃R³ is —SO₃H the —SO₃H group can beprotected by esterification.

Further Reaction Steps:

After the condensation with maleimidoalkylic acid, the 9-hydroxymethylcan be deprotected by removing the protecting group (PG) to yieldcompounds of formula 2. Deprotection is preferably performed with BF₃,e.g. BF₃Et₂O (Boron trifuorid etherate).

MAL-FMS-OSU or its derivatives may be synthesized by reacting a compoundof formula 2 with N-hydroxysuccinimide or its derivatives, such asN,N′-Disuccinimidyl carbonate. Reaction conditions for the formation ofa succinimidyl-ester are well known in the art. A succinimidyl-modifiedcompound for formula 2 can be further modified by reaction withSH-polymers and subsequently reacted with amino groups of peptide orprotein drugs to yield conjugates of peptide or protein drugs having ahydrolysable linker containing a polymer.

The protocol exemplified above yields a compound of formula 1:

wherein PG is a protecting group and at least one of position 1, 2, 3,4, 5, 6, 7 or 8 is bound to radical Y.

Y is a N-maleimidyl-containing moiety.

Compounds of formula 1, which are substituted with at least one radicalY may also be bound to radical X in at least one of the availableposition 1, 2, 3, 4, 5, 6, 7 or 8.

-   -   X is —SO₃—R³.    -   R³ is independently selected from the group consisting of        hydrogen, (C₁-C₈)-alkyl and (C₁-C₈)-alkyl-R⁴.    -   “C₁-C₈-alkyl” refers to monovalent alkyl groups having 1 to 8        carbon atoms. This term is exemplified by groups such as methyl,        ethyl, propyl, butyl, hexyl and the like. Linear and branched        alkyls are included.

R⁴ is a polymer. Examples of polymers are poly(ethylene glycol) (PEG),polysialic acid (PSA), hydroxyalkyl starch (HAS) and the like.

In another embodiment the invention relates to a compound of formula 1,wherein PG is a silyl group. Examples of a silyl group aretrimethylsilyl, triethylsilyl or t-butyldiphenylsilyl.

In another embodiment PG is a tert-butyldimethylsilyl group.

In one embodiment Y is:

R¹ is at each occurrence independently a (C₁-C₈)-alkyl.

In one embodiment R¹ is at each occurrence independently selected fromthe group consisting of methyl, ethyl, propyl, butyl, and hexyl.

R² is independently selected from the group consisting of —C(O)NR—,—C(O)NR—(C₁-C₈)-alkyl-NR—, —NRC(O)— and —NRC(O)—(C₁-C₈)-alkyl-NR,wherein R is independently either hydrogen or C₁-C₈-alkyl.

In one embodiment R² is —C(O)NH—.

In another embodiment R² is —NHC(O)—.

In one embodiment the compound of formula 1 is bound to radical Y in atleast one of position 1, 2, 3 or 4.

In another embodiment the compound of formula 1, which is bound toradical Y in at least one of position 1, 2, 3, or 4 and is further boundto radical X in at least one of position 5, 6, 7, or 8.

In another embodiment the compounds of formula 1, which are substitutedwith at least one radical Y in at least one of position 2 or 3 may alsobe bound to radical X in at least one of position 7 or 8.

In another embodiment the compound of formula 1, which are substitutedwith at least one radical Y, radical X is bound to position 7.

In another embodiment the compounds of formula 1 are bound to radical Yin positions 2 and 7.

In another embodiment the compounds of formula 1 are bound to radical Yand radical X in positions 2 and 7, respectively.

In another embodiment the compound of formula 1 is:

The present invention is illustrated by the following examples withoutbeing limited thereto.

EXAMPLES

MAL-Fmoc-OSu molecules can be synthesised according to the followingprotocol (Examples 1-8):

Example 1 Synthesis of 2-(Boc-amino)fluorene (Albericio et al., SynthCommun. 31, 225-32 (2001))

A suspension of 2-Aminofluorene (14.4 g, 79.7 mmol) was prepared in amixture of 145 ml dioxane-H₂O (2:1/V:V), cooled with an ice bath and42.5 ml 2 N NaOH under gentle stirring. Boc₂O (19.1 g, 87.7 mmol, 1.1equiv) was then added and the stirring was continued at 25° C. Theevolution of the reaction was followed by TLC [R_(f)=0.75 for2-(Boc-amino) fluorene. CHCl₃-MeOH—HOAc (95:5:3)] and the pH wasmaintained between 9-10 by addition of 2 N NaOH. After 24 h TLC analysisshowed the presence of 2-aminofluorene [R_(f)=0.60, CHCl₃-MeOH—HOAc(95:5:3)], so another 5.2 g Boc₂O (23.8 mmol, 0.3 equiv) were added andthe reaction was continued for additional 3 h until the totaldisappearance of the starting product. The suspension was acidified with1 M KHSO₄ to pH 3. The solid was filtered and washed with 30 ml coldH₂O, 30 ml dioxane-H₂O (2:1), 30 ml hexane and dried in vacuum. Theproduct, a pale yellow powder (30.1 g, 90% yield) was shown to be pureby TLC [R_(f)=0.75; CHCl₃/MeOH/HOAc 95:5:3], and characterized by NMR.

¹H NMR (200 MHz/DMSO) δ=9.45 (1H; s; NH); 7.84-7.77 (3H; m; H1, H4, H5);7.59-7.17 (4H; m; H2, H6-H8); 3.86 (2H; s; CH₂); 1.49 (9H, s, t-Bu)

¹³C NMR (50 MHz/DMSO) δ=152.8 (Amid-C); 143.8 (C9a); 142.6 (C8a); 141.2(C4b); 138.7 (C2); 135.2 (C4a); 126.7 (C6); 125.8 (C7); 124.9 (C8);120.0 (C4); 119.2 (C5); 116.9 (C3); 114.8 (C1); 79.02 (CH₂); 39.5 (Cqt-Bu); 28.2 (3×CH₃ t-Bu)

Example 2 Synthesis of 9-Hydroxymethyl-2-(Boc-amino) fluorene (Albericioet al., Synth Commun. 31, 225-32 (2001))

A solution of 2-(Boc-amino)fluorene (13.49 g, 47.9 mmol) in 140 ml dryTHF (freshly distilled from sodium) was carefully added to a suspensionof 6.3 g 60% NaH (160 mmol, 3.3 equiv) in 20 ml dry THF under argonatmosphere. Gas evolution and spontaneous warming were observed. Afterthe complete addition of 2-(Boc-amino)fluorene the reaction mixture wasstirred for 1 h at 40° C. Then the reaction mixture was cooled to roomtemperature and 9.7 ml ethyl formiate (120 mmol, 2.5 equiv) were slowlyadded to avoid vigorous hydrogen bubbling. The initially thick, lightbrown suspension rapidly clarified to a dark brown solution uponaddition of ethyl formiate and was stirred for 1 h. The evolution of thereaction was followed by TLC [Rf=0.52 for the intermediate product,CHCl₃-MeOH—HOAc (95:5:3)]. The reaction was quenched with ice chips and100 ml H₂O and the organic solvent was removed by rotary evaporation. Tothe aqueous phase 10 ml 2 N NaOH were added and it washed with 3×50 mldiethylether, cooled in an ice bath and acidified with 25 ml glacialHOAc until pH 5. The off-white precipitate that then appeared wasdissolved in 300 ml EtOAc. The aqueous phase was extracted with 50 mlEtOAc and the organic phase was washed with 2×75 ml sat. NaHCO₃ and 1×75ml brine and dried over Na₂SO₄. The solvent was eliminated under reducedpressure.

9-Formyl-2-(Boc-amino)fluorene was suspended in 100 ml methanol and 2.0g NaBH₄ [52.9 mmol, 1.1 equiv with respect to the starting2-(Boc-amino)fluorene] was added portion wise. The suspension thatrapidly cleared up was magnetically stirred until the starting productdisappeared for 4 h at room temperature [TLC, R_(f)=0.57, PE:MTBE(1:2)]. The reaction mixture was diluted with 300 ml H₂O and acidifiedwith 15 ml glacial HOAc to pH 5.0, and the precipitate was directlydissolved in 150 ml EtOAc. The organic phase was washed with 3×50 mlsat. NaHCO₃ and 1×50 ml brine and dried over anhydrous Na₂SO₄. Thesolvent was rotary evaporated to give a solid, which could be usedwithout further purification. The product was analyzed by NMR (13.1 g,88% yield).

¹H NMR (200 MHz/DMSO) δ=9.42 (1H; s; NH); 7.87 (1H; s; H1); 7.79-7.66(2H; m; H4, H5); 7.61 (1H; d; J=7.71 Hz; H8); 7.48-7.15 (3H, m, H3, H6,H7;); 5.07 (1H, t, J=4.80 Hz; OH); 4.00-3.88 (1H; m; H9); 3.82-3.63 (2H;m; CH₂); 1.49 (9H; s; t-Bu)

¹³C NMR (50 MHz/DMSO) δ=152.88 (Amid-C); 146.05 (C9a); 144.90 (C8a);140.81 (C4b); 138.64 (C2); 134.90 (C4a); 127.13 (C6); 125.85 (C7);125.05 (C8); 119.93 (C4); 119.16 (C5); 117.47 (C3); 115.24 (C1); 79.00(Cq t-Bu); 63.85 (CH₂); 50.19 (C9); 28.20 (3×CH₃ t-Bu)

Example 3 Synthesis of 9-Hydroxymethyl-2-amino fluorene

13.0 g 9-Hydroxymethyl-2-(Boc-amino)fluorene was dissolved in 110 mlacetonitrile and stirred under reflux. 42 ml 2 N HCl (2.0 equiv, 84mmol) was added drop-wise. The reaction mixture was stirred under refluxfor 45 min. The reaction mixture was cooled to room temperature and thereaction was monitored by TLC [R_(f)=0.1 PE-MTBE (1:2)]. The solvent waspartially eliminated by rotary evaporation and the residue was dissolvedin 70 ml 2 N HCl. The solution was carefully washed with 2×50 ml MTBE.The aqueous phase was adjusted to pH 9 by Na₂CO₃ and extracted with 2×70ml EtOAc. The organic phase was washed with 50 ml brine and dried overNa₂SO₄. The solvent was eliminated by rotary evaporation. The productwas used without further purification. The structural identity wasverified by NMR (8.76 g, 99% yield).

NMR (200 MHz/DMSO) δ=7.69-7.40 (3H; m; 3×Ar—H); 7.37-7.02 (2H; m;2×Ar—H); 6.87 (1H; s; Ar—H); 6.68 (1H; d; J=8.34 Hz; Ar—H); 5.19 (2H; s;NH₂); 5.03 (1H; t; J=4.93 Hz; OH); 3.93-3.58 (3H; m; H9, CH₂)

¹³C NMR (50 MHz/DMSO) δ=148.36 (Ar-Cqu); 146.81 (Ar-Cqu); 143.97(Ar-Cqu); 141.87 (Ar-Cqu); 129.03 (Ar-Cqu); 126.92 (Ar—CH); 124.85(Ar—CH); 124.24 (Ar—CH); 120.40 (Ar—CH); 117.81 (Ar—CH); 113.00 (Ar—CH);110.76 (Ar—CH); 64.27 (CH₂); 49.90 (CH)

Example 4 Synthesis of tert-Butyldimethylsiloxy-9-methyl-2-aminofluorene

5.91 g Imidazole (86.8 mmol, 2.1 equiv) was dissolved in 24 ml dry DMFand stirred 10 min in an iced bath under argon atmosphere. 7.47 gtert-Butyldimethylsilyl chloride (49.6 mmol, 1.2 equiv) dissolved in dryDMF was added. After 15 min stirring on ice 8.73 g9-Hydroxymethyl-2-amino fluorene (41.3 mmol) dissolved in 40 ml dry DMFwas added drop wise under cooling and argon atmosphere. The reaction wascontinued 15 min on ice and then at room temperature. The reaction wasmonitored by TLC [title product R_(f)=0.6, PE-MTBE (1:2)]. After 2 hoursthe starting product [R_(f)=0.1 PE-MTBE (1:2)] had disappeared and thereaction mixture was diluted with 400 ml CH₂Cl₂ and 100 ml 5% NaHCO₃ wasadded. The organic phase was washed with 5×200 ml H₂O and dried overNa₂SO₄. CH₂Cl₂ was eliminated by rotary evaporation and DMF waseliminated by azeotropic distillation with toluene. The residual brownoil (13.4 g, 99% yield) was analyzed by NMR and was used without furtherpurification.

¹H NMR (200 MHz/DMSO) δ=7.67-7.40 (3H; m; 3×Ar—H); 7.34-7.00 (2H; m;2×Ar—H); 6.81 (1H; s; 1×Ar—H); 6.59 (1×; dd; J=8.02 Hz & 1.83 Hz;1×Ar—H); 5.19 (2H; s; NH₂); 3.97-3.76 (2H; m; CH₂); 3.75-3.57 (1H; m;CH); 0.88 (9H; s; 3×CH₃); 0.03 (6H; s; 2×CH₃)

¹³C NMR (50 MHz/DMSO) δ=148.40 (Ar-Cqu); 145.81 (Ar-Cqu); 143.67(Ar-Cqu); 141.88 (Ar-Cqu); 129.08 (Ar-Cqu); 127.10 (Ar—CH); 124.97(Ar—CH); 124.16 (Ar—CH); 120.47 (Ar—CH); 117.87 (Ar—CH); 113.22 (Ar—CH);110.58 (Ar—CH); 66.04 (CH₂—OH); 49.60 (C9); 25.88 (3×CH₃; t-Bu); 18.04(Cqu; t-Bu), −5.04 (2CH₃; Si—CH₃)

Example 5 Synthesis oftert-Butyldimethylsiloxy-9-methyl-2-(amino-3-maleimidopropionate)fluorene

To a solution of 13.5 g 9-tert-Butyldimethylsiloxymethyl-2-aminofluorene(41.5 mmol) in dry THF (freshly distilled from sodium) 9.42 gN,N′-dicyclohexylcarbodiimide (75.7 mmol, 1.1 equiv) and 651 mg1-hydroxybenzotriazole (4.8 mmol, 0.1 equiv) were added. 13.5 g (41.5mmol, 1.1 equiv) 3-maleimidopropionic acid was dissolved in 50 ml dryTHF and added dropwise. The reaction mixture was stirred at roomtemperature over night under argon atmosphere and the product formationwas monitored by TLC [starting material R_(f)=0.6, title productR_(f)=0.18, PE-MTBE (1:2)]. As soon as the starting material could notbe detected, dicyclohexylurea was filtered out and THF was eliminated byrotary evaporation. The residual solid was dissolved in 200 ml CH₂Cl₂,washed with 50 ml 5% NaHCO₃ and 50 ml brine and dried over Na₂SO₄. Thebrown crystals were digerated in 20 ml MTBE. After filtration theresidue was washed with small portions of MTBE until the washingsolution maintained colorless. The yellow crystals (10.5 g, 53% yield)were analyzed by NMR.

¹H NMR (200 MHz/DMSO) δ=10.06 (1H; s; NH); 7.92 (1H; s; H1); 7.82-7.70(2H; m; H4 & H5); 7.62 (1H; d; J=7.20 Hz; H3); 7.49 (1H; d; J=8.08 Hz;H8); 7.41-7.18 (2H; m; H6 & H7); 7.03 (2H; s; 2×Mal-CH); 4.02 (1H; t;J=6.63 Hz; H9); 3.95-3.66 (4H; m; Prop-CH₂—N & CH₂—OTBDMS); 2.61 (2H; t;J=7.07 Hz; Prop-CH₂—C═O); 0.84 (9H; s; 3×t-Bu CH₃); 0.10-0.56 (6H; m;2×CH₃—Si)

¹³C NMR (50 MHz/DMSO) δ=170.81 (C═O Mal); 168.30 (C═O Amid); 145.12(C9a); 144.62 (C8a); 140.68 (C4b); 138.05 (C2); 136.02 (C4a); 134.62(2×CH Mal); 127.34 (C6); 126.02 (C7); 125.15 (C8); 119.99 (C4); 119.37(C5); 118.63 (C3); 116.31 (C1); 65.54 (CH₂OTBDMS); 49.85 (C9); 35.01(Prop-CH₂—N); 33.91 (Prop-CH₂—CO); 25.84 (3×CH₃ (t-Bu); 18.01 (Cqut-Bu); −5.40 (CH₃—Si); −5.44 (CH₃—Si)

Example 6 Synthesis of9-Hydroxymethyl-2-(amino-3-maleimidopropionate)fluorene

10.3 g tert-Butyldimethylsiloxy-9-methyl-2-(amino-3-maleimidopropionate)fluorene (21.6 mmol) was dissolved in 230 ml CH₂Cl₂ under argonatmosphere. 35 ml boron trifluoride etherate were added drop-wise over30 min. The reaction was monitored by TLC [starting material R_(f)=0.6,title product R_(f)=0.38, CH₂Cl₂-Methanol (10:1)]. As soon as thestarting material had disappeared the solution was hydrolyzed with sat.NaHCO₃ solution. The resulting crystals were filtered. The organic phaseof the mother liquor was evaporated. This residue and the filter cakewere re-dissolved in 250 ml EtOAc and 120 ml 5% NaHCO₃. The organicphase was washed with 1×50 ml 5% NaHCO₃, 50 ml H₂O and 50 ml brine anddried over Na₂SO₄. The solvent was eliminated by rotary evaporation. Thestructure of the product was verified by NMR and mass spectroscopy.

¹H NMR (200 MHz/DMSO) δ=10.09 (1H; s; NH); 7.88 (1H; s; H1); 7.82-7.69(2H; m; H4 & H5); 7.66-7.52 (2H; m; H3 & H8); 7.41-7.17 (2H; m; H6 &H7); 7.03 (2H; s; 2×Mal-CH); 5.09 (1H; t; J=4.93 Hz; OH); 5.08 (1H; t;J=6.06 Hz; H9); 3.97-3.53 (4H; m; Prop-CH₂—N& CH₂—OH); 2.61 (2H; t;J=6.82 Hz; Prop-CH₂—C═O)

¹³C NMR (50 MHz/DMSO) δ=170.82 (C═O Mal); 168.38 (C═O Amid); 146.00(C9a); 144.89 (C8a); 140.63 (C4b); 138.05 (C2); 135.92 (C4a); 134.62(2×CH Mal); 127.16 (C6); 126.09 (C7); 125.03 (C8); 119.93 (C4); 119.33(C5); 118.39 (C3); 116.36 (C1); 63.86 (CH₂OH); 50.14 (C9); 35.07(Prop-CH₂—N); 33.92 (Prop-CH₂—CO)

ESI-MS: found: (M+Na)⁺: 385.2; calculated: (M+Na)⁺: 385

Example 7 Synthesis of9-Hydroxymethyl-2-(amino-3-maleimidopropionate)fluoreneN-hydroxysuccinimidyl carbonate

From a solution of 1.8 g pyridine in 7.3 ml abs THF 1.7 ml (5.1 mmol)were added drop wise to a stirred solution of 0.93 g9-Hydroxymethyl-2-(amino-3-maleimidopropionate) fluorene (2.6 mmol) and1.1 g triphosgene (3.6 mmol, 1.4 equiv) in 75 ml dry THF (freshlydistilled from sodium). After 40 min the precipitated pyridinehydrochloride salt was filtered out over celite, and the THF was removedby rotary evaporation. The oil obtained was dissolved in 75 ml drytetrahydrofuran with 1.1 g N-hydroxysuccinimide (13.6 mmol, 5.3 equiv).2.6 ml of the pyridine solution (8.2 mmol) were then added, and thesolution was stirred for 40 min. Some additional precipitated pyridinehydrochloride salt was filtered out over celite, and the THF was removedby rotary evaporation. The oil obtained was dissolved in 70 mlchloroform and washed with 4×40 ml 0.1N HCl, 3×50 ml of an aqueous 5%NaHCO₃ solution, then with 1×40 ml water, 40 ml brine and dried overNa₂SO₄. The chloroform was removed by rotary evaporation. Structuralidentity was verified by NMR and mass spectroscopy.

¹H NMR (500 MHz/CDCl₃) δ=8.35 (1H; s; NH); 8.00-7.95 (1H; m; H3);7.71-7.65 (2H; m; H4 & H5); 7.50-7.46 (1H; d; J=7.25 Hz; H8); 7.42-7.36(2H; m; H1 & H6); 7.30-7.25 (1H; m; H7); 6.69 (2H; s; 2×Mal-CH); 4.66(1H; dd; J=10.40 Hz & 5.99 Hz; H_(a)—CH₂O); 4.14 (1H; t; J=10.09 Hz;H_(b)—CH₂O); 4.27 (1H; dd; J=9.46 Hz & 5.99 Hz; H9); 3.95 (2H; t; J=7.09Hz; Prop-CH₂—N); 2.86 (4H; s; 2×Succ-CH₂); 2.80-2.54 (2H; m;Prop-CH₂—C═O)

¹³C NMR (125 MHz/CDCl₃) δ=170.53 (C═O Mal); 168.21 (C═O Amid); 151.41(C═O Carbonate); 143.79 (C2); 141.13 (C9a); 140.94 (C8a); 137.36 (C4b);137.11 (C4a); 134.20 (2×CH Mal); 128.33 (C6); 126.82 (C7); 124.62 (C8);120.63 (C4); 120.23 (C3); 119.96 (C5); 116.50 (C1); 72.67 (CH₂O); 46.49(C9); 35.62 (Prop-CH₂—N); 34.15 (Prop-CH₂—CO); 25.47 (2×Succ-CH₂)

ESI-MS (calculated): (M+H)⁺: 504; (M+Na)⁺: 526

ESI-MS (found): (M+H)⁺: 504.1; (M+Na)⁺: 526.1

Example 8 Synthesis of9-Hydroxymethyl-2-(amino-3-maleimidopropionate)-7-sulfo fluoreneN-hydroxysuccinimidyl carbonate

To a solution of 1.2 g 9-Hydroxymethyl-2-(amino-3-maleimidopropionate)fluorene N-hydroxysuccinimidyl carbonate (2.1 mmol) in 60 mltrifluoroacetic acid 7 ml chlorosulfonic acid was added. After 30 minthe reaction mixture was cooled to 4° C. and 350 ml cold diethyletherwas added. The precipitated product was filtered and washed twice withdiethylether and dried in vacuum.

Structural identity was verified by mass spectroscopy.

ESI-MS (found): (M+H)⁺: 583.9

ESI-MS (calculated): (M+H)⁺: 583

The synthesis of MAL2-Fmoc-OSu starting from 2,7 Diaminofluorene isillustrated in Example 9.

Example 9 Synthesis of9-Hydroxymethyl-2,7-Di-(amino-3-maleimidopropionate) fluoreneN-hydroxysuccinimidyl carbonate

9-Hydroxymethyl-2,7-Di-(amino-3-maleimidopropionate) fluoreneN-hydroxysuccinimidyl carbonate is prepared under the conditions asdescribed in Examples 1-8. The amino groups in 2,7-Diaminofluorene areprotected with BOC₂O as described by Albericio et al., Synth Commun. 31,225-32 (2001). Then the formyl-group is introduced in position 9 byreaction of Lithiumdiisopropylamide (LDA) and ethylformiate. Thealdehyde obtained is reduced with sodiumborhydride to the correspondingalcohol to form 9-Hydroxymethyl-2,7-di-(Boc-amino)fluorene. Subsequentlythe BOC protecting groups are cleaved with 2N HCl in CH₃CN and9-Hydroxymethyl-2,7-diaminofluorene is obtained. Then the OH-group isprotected by reaction with tert.-Butyldimethylsilylchloride as describedin Example 5 to formtert-Butyldimethylsiloxy-9-methyl-2,7-diaminofluorene. Then the reactionof the free amino groups with maleimidopropionic acid in the presence ofN,N′-dicyclohexylcarbodiimide and hydroxybenzotriazole is performed andtert-Butyldimethylsiloxy-9-methyl-2,7-di-(amino-3-maleimidopropionate)fluorene is obtained. After deprotection of the OH group in position 9with Boron trifluoride etherate9-Hydroxymethyl-2,7-di-(amino-3-maleimidopropionate) fluorene is formed.Finally the reaction with Triphosgene and N-hydroxysuccinimide iscarried out and 9-Hydroxymethyl-2,7-di-(amino-3-maleimidopropionate)fluorene N-hydroxysuccinimidyl carbonate is prepared.

1. A method of preparing a compound according to formula 1:

wherein PG is a protecting group and at least one of position 1, 2, 3,4, 5, 6, 7 or 8 is bound to radical Y; Y is a radical containing aN-maleimidyl-moiety; and at least one of an available position 1, 2, 3,4, 5, 6, 7 or 8 is optionally bound to radical X; X is —SO₃—R₃; R³ isindependently selected from the group consisting of hydrogen,(C₁-C₈)-alkyl and (C₁-C₈)-alkyl-R⁴; R⁴ is a polymer; and the compound offormula 1 is crystalline; said method comprises reacting the9-hydroxymethyl group of a compound of formula 2:

wherein at least one of position 1, 2, 3, 4, 5, 6, 7, or 8 is bound toan amine, with a protecting group reagent introducing PG, andsubsequently reacting said compound with an N-maleimidyl reagent.
 2. Themethod according to claim 1, wherein said protecting reagent is a silylhalide.
 3. The method according to claim 2, wherein said silyl halide istert-butyldimethylsilyl chloride (t-BDMS-Cl).
 4. The method according toclaim 3, wherein said reaction with tBDMS-Cl is performed with imidazolein dimethylformamide.
 5. The method according to claim 4, wherein saidN-maleimidyl-reagent is maleimidoalkylic acid.
 6. The method accordingto claim 1, wherein PG is a silyl derivative.
 7. The method according toclaim 6, wherein PG is a is tert-butyldimethylsilyl group.
 8. The methodaccording to claim 7, wherein at least one of an available position 1,2, 3, 4, 5, 6, 7 or 8 is bound to radical X.
 9. The method according toclaim 7 wherein Y is

wherein R¹ is at each occurrence independently a (C₁-C₈)-alkyl, and R²is independently selected from the group consisting of —C(O)NR—,—C(O)NR—(C₁-C₈)-alkyl-NR—, —NRC(O)— and —NRC(O)—(C₁-C₁)-alkyl-NR,wherein R is independently either hydrogen or C₁-C₈-alkyl.
 10. Acompound of the formula 1,

wherein PG is a protecting group and at least one of position 1, 2, 3,4, 5, 6, 7 or 8 is bound to radical Y; and at least one of an availableposition 1, 2, 3, 4, 5, 6, 7 or 8 is optionally bound to radical X; Y isa N-maleimidyl-containing moiety; X is —SO₃R³; R³ is independentlyselected from the group consisting of hydrogen, (C₁-C₈)-alkyl and(C₁-C₈)-alkyl-R⁴; and the compound of formula 1 is crystalline.
 11. Thecompound according to claim 10, wherein PG is a silyl protecting group,Y is:

R¹ is at each occurrence independently a (C₁-C₈)-alkyl; and R² isindependently selected from the group consisting of —C(O)NR—,—C(O)NR—(C₁-C₈)—alkyl-NR—, —NRC(O)— and —NRC(O)—(C₁-C₈)-alkyl-NR—,wherein R is independently either hydrogen or C₁-C₈-alkyl.
 12. Thecompound according to claim 11, wherein PG is a t-butyldimethylsilylgroup.